Cobas amplicor monitor 2.0 assay

Plasma HIV RNA levels were measured using the NASBA/NuciSens HIV RNA assay (BioMerieux, Durham, NC, USA), in laboratories certified by the NIH National Institute of Allergy and Infectious Diseases Virology Quality Assurance Certification Program. HCV and HBV serological markers were performed using standard commercial assays and included hepatitis C antibody by EIA 3.0 (Ortho-Clinical Diagnostics, Raritan, NJ, USA) and hepatitis B surface antigen (HBsAg) (Abbott Laboratories, Abbott Park, IL, USA). HCV RNA levels were measured by the COBAS Amplicor Monitor 2.0 assay (Roche Diagnostics, Branchburg, NJ, USA) with a linear range of 600–700 000 IU/mL, or COBAS TaqMan (Roche Diagnostics), with a linear range of 10–2.0 × 10⁸ IU/mL. Genotyping for rs368234815 (ss469415590) was performed at the Laboratory of Translational Genomics National Cancer Institute with custom TaqMan allelic discrimination genotyping assays, as previously described [14 (link)]. IFNL3/IFNL4 SNPs were genotyped as part of the WIHS genomewide association study using the IlluminaOmni2.5-quad beadchip (Illumina Inc, San Diego, CA, USA). Ancestry informative marker SNPs were selected using Helix Tree (Golden Helix, Bozeman, MT, USA).

Plasma HIV-1 RNA was measured by isothermal nucleic acid sequence-based amplification (NASBA/Nuclisens; Organon Teknika Corp., Durham, NC, USA) with a lower limit of detection (LLD) of 80 copies/ml prior to October 1, 2008 and then by COBAS Taqman HIV-1 assay with LLD of 48 or 20 copies/mL, based on assay kit detection performance. Lymphocyte subsets were quantified using standard flow cytometric methods in laboratories participating in the NIH/NIAID Flow Cytometry Quality Assessment Program.^{42 (link)} HCV RNA was measured on frozen repository specimens for all women who tested HCV antibody-positive at WIHS enrollment using either the COBAS Amplicor Monitor 2.0 assay or the COBAS Taqman assay (Roche Diagnostics, Branchburg, NJ, USA).^{43 (link)}